Microorganisms and processes for the production of isoprene

ABSTRACT

The present invention provides a novel biosynthetic pathway for the production of isoprene from 3-methyl-2-buten-1-ol or 2-methyl-3-buten-2-ol. Further embodiments provide non-naturally occurring microorganism that have been modified to produce isoprene from 3-methyl-2-buten-1-ol or 2-methyl-3-buten-2-ol and methods of producing isoprene using said microorganism.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser. No. 13/894,744, filed on May 15, 2013, which claims the benefit of U.S. Provisional Application No. 61/776,485, filed on Mar. 11, 2013 and U.S. Provisional Application No. 61/688,514, filed on May 16, 2012. The entire teachings of the above applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present disclosure generally relates to the use of a non-naturally occurring microorganism for the production of isoprene. More specifically, the present disclosure relates to non-naturally occurring microorganisms that have been modified to express enzymes that enable the production of isoprene from different alcohols, in particular 3-methyl-2-buten-1-ol or 2-methyl-3-buten-2-ol.

BACKGROUND OF THE INVENTION

Currently, many high-value chemicals or fuels are typically manufactured by thermochemical processes from hydrocarbons, including petroleum oil and natural gas. Also, high value chemicals may be produced as “by-products” during the processing of crude oil into usable fractions. For example, isoprene has typically been produced during the catalytic cracking of crude oil fractions. However, recently catalytic cracker users have shifted their focus from crude oil to natural gas, resulting in a reduced source of the four and five carbon chain molecules that are found in crude oil, but not natural gas.

Being a short-chain carbon molecule, isoprene is a useful starting material for synthesizing a variety of chemicals. Isoprene may be used as a monomer or co-monomer for the production of higher value polymers. Examples of chemicals that can be produced using isoprene include polyisoprene, polybutylene, styrene-isoprene-styrene block co-polymers, and others. An example of an industry that uses isoprene is the synthetic rubber industry.

Given the increasing demand, decreasing supply and the many uses of isoprene, a new method of isoprene production is desired. Also, as the concerns of energy security, increasing oil and natural gas prices, and global warming escalate, the chemical production industry is seeking ways to replace chemicals made from non-renewable feedstocks with chemicals produced from renewable feedstocks using environmentally friendly practices.

The biological production of isoprene has been studied since the 1950s (Sharkey, T. D. 2009. The Future of Isoprene Research. Bull. Georg. Natl. Acad. Sci. 3: 106-113). Although many different organisms are known to emit isoprene, so far the biochemical pathway for isoprene production has only been elucidated in a few plant species. In plants, it appears that isoprene is produced in the chloroplast or other plastids from dimethylallyl diphosphate, also referred to herein as dimethylallyl pyrophosphate (DMAPP), in a single step by isoprene synthase, a nuclearly encoded enzyme that is routed to the plastid by a plastid targeting signal sequence. The isoprene synthases generally have a high Michaelis-Menten constant (K_(m)), typically 1 millimolar or higher, and thus require high concentrations of dimethylallyl diphosphate to function efficiently.

Although microbes that naturally produce isoprene are known in the art (Kuzma, J., Nemecek-Marshall, M., Pollock, W. H., and R. Fall. 1995. Bacteria produce the volatile hydrocarbon isoprene. Curr. Microbiol. 30: 97-103; Wagner, W. P., Nemecek-Marshall, M., and R. Fall. 1999. Three distinct phases of isoprene formation during growth and sporulation of Bacillus subtilis. J. Bact. 181: 4700-4703; Fall, R. and S. D. Copley. 2000. Bacterial sources and sinks of isoprene, a reactive atmospheric hydrocarbon. Env. Microbiol. 2: 123-130; Xue, J., and B. K. Ahring. 2011. Enhancing isoprene production by the genetic modification of the 1-deoxy-D-xylulose-5-phosphate pathway in Bacillus subtilis. Appl. Env. Microbiol. 77: 2399-2405), the mechanism of isoprene production is unknown and the levels of isoprene production are relatively low. Several non-naturally occurring microorganisms have been engineered to produce isoprene, e.g., U.S. patent application Ser. No. 12/335,071, wherein isoprene production requires an isoprene synthase. For efficient function of isoprene synthase, high intracellular levels of dimethylallyl diphosphate are required; however, high levels of intracellular dimethylallyl diphosphate are also toxic to the cells, retarding growth and reducing the rates and yields of isoprene production (Martin, V. J. J., Pitera, D. J., Withers, S. T., Newman, J. D. and J. D. Keasling. 2003. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotech. 21: 796-802; Withers, S. T., Gottlieb, S. S., Lieu, B., Newman, J. D., and J. D. Keasling. 2007. Identification of isopentenol biosynthetic genes from Bacillus subtilis by a screening method based on isoprenoid precursor toxicity. Appl. Env. Microbiol. 73: 6277-7283; Sivy, T. L., Fall, R., and T. N. Rosentiel. 2011. Evidence of isoprenoid precursor toxicity in Bacillus subtilis. Biosci. Biotechnol. Biochem. 75: 2376-2383). The problems associated with the direct chemical conversion of DMAPP to isoprene by isoprene synthases limits the potential for the biological production of commercially relevant amounts of isoprene.

Thus, there is a need for microorganisms and processes for the more efficient biological production of isoprene.

SUMMARY OF THE INVENTION

Embodiments of the present invention generally provide enzymes, non-naturally occurring microorganisms, and methods of producing isoprene.

Embodiments of the invention provide non-naturally occurring microbial organisms, i.e., microorganisms that include a biosynthetic isoprene pathway. The microorganisms include an exogenous nucleic acid encoding an enzyme of the biosynthetic pathway. The enzyme is a 2-methyl-3-buten-2-ol dehydratase, and the biosynthetic pathway is expressed at a sufficient level to produce isoprene. The biosynthetic pathway may further comprise a 2-methyl-3-buten-2-ol isomerase. The 2-methyl-3-buten-2-ol isomerase may be part of a bi-functional enzyme that also has the 2-methyl-3-buten-2-ol dehydratase activity. An example of such a bi-functional enzyme is a linalool dehydratase-isomerase. The microorganism may further comprise a 3-methyl-2-buten-1-ol synthase.

In another embodiment, a non-naturally occurring microorganism comprising a biosynthetic isoprene pathway is provided, wherein the microorganism comprises an exogenous nucleic acid encoding an enzyme of the biosynthetic isoprene pathway, 2-methyl-3-buten-2-ol dehydratase. The pathway further comprises a 2-methyl-3-buten-2-ol synthase, and the pathway is expressed at a sufficient level to produce isoprene.

In one embodiment, the present invention provides for a non-naturally occurring microorganism comprising at least one or more exogenous nucleic acids encoding one or more enzymes of an isoprene biosynthetic pathway, wherein the one or more enzymes of an isoprene biosynthetic pathway are expressed in sufficient amounts to produce isoprene, said isoprene biosynthetic pathway comprising a 3-methyl-2-buten-1-ol synthase, a 2-methyl-3-buten-2-ol isomerase, and a 2-methyl-3-buten-2-ol dehydratase.

In another embodiment, the present invention provides for a non-naturally occurring microorganism comprising at least one or more exogenous nucleic acids encoding one or more enzymes of an isoprene biosynthetic pathway, wherein the one or more enzymes of an isoprene biosynthetic pathway are expressed in sufficient amounts to produce isoprene, said isoprene biosynthetic pathway comprising a 2-methyl-3-buten-2-ol synthase and a 2-methyl-3-buten-2-ol dehydratase.

In an additional embodiment, the present invention provides for a method of producing isoprene, the method comprising the steps of culturing a non-naturally occurring microbial organism comprising at least one or more exogenous nucleic acids encoding one or more enzymes of an isoprene biosynthetic pathway, wherein the one or more enzymes of an isoprene biosynthetic pathway are expressed in sufficient amounts to produce isoprene, in a suitable culture medium containing a carbon source under conditions such that the non-naturally occurring microorganism converts at least a part of the carbon source to isoprene, and recovering the isoprene.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features of the present disclosure can be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

FIG. 1 shows an isoprene biosynthetic pathway comprising a 3-methyl-2-buten-1-ol synthase, a 2-methyl-3-buten-2-ol isomerase, and a 2-methyl-3-buten-2-ol dehydratase.

FIG. 2 shows an isoprene biosynthetic pathway comprising a 2-methyl-3-buten-2-ol synthase and a 2-methyl-3-buten-2-ol dehydratase.

FIG. 3 shows an E coli-codon-optimized nucleic acid sequence (SEQ ID NO: 1), including an artificial ribosome binding site and an amino-terminal 6-histidine epitope tag, for the linalool dehydratase isomerase of Castellaniella defragrans strain 65Phen.

FIG. 4 shows an E coli-codon-optimized nucleic acid sequence (SEQ ID NO: 2), including an artificial ribosome binding site and an amino-terminal 6-histidine epitope tag, for strawberry alcohol acyltransferase.

FIG. 5 shows a gas chromatogram of a 1 ml sample of the headspace of a 20-ml vial containing 1 mM 3-methyl-2-buten-1-ol dissolved in Luria Bertani broth. Peak 1 is 3-methyl-2-buten-1-ol, with a retention time of 3.96 minutes.

FIG. 6 shows a gas chromatogram of a 1 ml sample of the headspace of a 20 ml vial containing E. coli strain BL21 harboring plasmid pJ404-LDI cultured overnight on 1 mM 3-methyl-2-buten-1-ol in Luria Bertani broth supplemented with 100 μg/ml ampicillin. Peak 1 is 3-methyl-2-buten-1-ol, with a retention time of 3.96 minutes. Peak 2 is 2-methyl-3-buten-2-ol with a retention time of 2.96 minutes. Peak 3 is isoprene, with a retention time of 2.49 minutes.

FIG. 7 shows a gas chromatogram of a 1 ml sample of the headspace of a 20 ml containing 1 mM 2-methyl-3-buten-2-ol dissolved in Luria Bertani broth. Peak 1 is 2-methyl-3-buten-2-ol with a retention time of 2.96 minutes.

FIG. 8 shows a gas chromatogram of a 1 ml sample of the headspace of a 20-milliliter vial containing E. coli strain BL21 harboring plasmid pJ404-LDI cultured overnight on 1 mM 2-methyl-3-buten-2-ol in Luria Bertani broth supplemented with 100 μg/ml ampicillin. Peak 1 is 2-methyl-3-buten-2-ol with a retention time of 2.96 minutes. Peak 2 is isoprene with a retention time of 2.49 minutes.

FIG. 9 shows the results of GC/MS analysis of authentic isoprene.

FIG. 10 shows the results of GC/MS analysis of the peak at 2.49 minutes from Example 1, verifying the identity of the peak as isoprene.

FIG. 11 shows an E coli-codon-optimized nucleic acid sequence (SEQ ID NO: 3), including an artificial ribosome binding site, for the Bacillus subtilis yhfR gene.

FIG. 12 shows an E coli-codon-optimized nucleotide sequence (SEQ ID NO: 4), including artificial binding sites and restriction endonuclease sites for subcloning, for a synthetic operon encoding FaNES 1 from strawberry and idi from H. pluvialis.

FIG. 13 shows a gas chromatogram of a 1 ml sample of the headspace of a 20 ml vial containing 1 mM linalool dissolved in Luria Bertani broth. Peak 1 is linalool, with a retention time of 8.8 minutes.

FIG. 14 shows a gas chromatogram of a 1 ml sample of the headspace of a 20 ml vial containing 1 mM 2-methyl-3-buten-2-ol dissolved in Luria Bertani broth under the same column conditions used to detect linalool. Peak 1 is 2-methyl-3-buten-2-ol, with a retention time of 4.8 minutes.

FIG. 15 shows a gas chromatogram of a 1 ml sample of headspace of a 20 ml vial containing E. coli BL21 harboring plasmid pJ401-NES1-idi cultured for 24 hours in Luria Bertani broth supplemented with 50 μg/ml kanamycin and 100 μM IPTG. Peak 1 is linalool, with a retention time of 8.8 minutes. Peak 2 is 2-methyl-3-buten-2-ol, with a retention time of 4.8 minutes. The peak at 4.75 minutes has been identified as 2-butanone.

FIG. 16 shows a gas chromatogram of a 1 ml sample of headspace of a 20 ml vial containing E. coli BL21 harboring plasmid pJ404-SAAT cultured for 24 hours in Luria Bertani broth supplemented with 100 μg/ml ampicillin and 100 μM IPTG. Peaks corresponding to linalool and 2-methyl-3-buten-2-ol are absent. The peak at 4.75 minutes has been identified as 2-butanone.

FIG. 17 shows the results of GC/MS analysis of authentic linalool.

FIG. 18 shows the results of GC/MS analysis of the peak at 8.8 minutes from FIG. 15, verifying the identity of the peak as linalool.

FIG. 19 shows the results of GC/MS analysis of authentic 2-methyl-3-buten-2-ol.

FIG. 20 shows the results of GC/MS analysis of the peak at 4.9 minutes from FIG. 15, verifying the identity of the peak as 2-methyl-3-buten-2-ol.

FIG. 21 shows the DNA sequence (SEQ ID NO: 5) of plasmid pGA31R-mcs.

FIG. 22 shows the DNA sequence (SEQ ID NO: 6) of plasmid pGS31R-mcs.

FIG. 23 shows the E coli-codon-optimized sequence (SEQ ID NO: 7) of the mvaE and mvaS genes of Enterococcus faecalis ATCC 700802, including incorporated ribosome binding sites and flanking restriction endonuclease sites used in subsequent cloning steps.

FIG. 24 shows the E coli-codon-optimized sequence (SEQ ID NO: 8) of the synthetic operon encoding the mevalonate kinase gene of Methanocaldococcus jannaschi, the phosphomevalonate kinase gene of Enterococcus faecalis ATCC 700802, the mevalonate diphosphate decarboxylase gene of Saccharomyces cerevisiae S288C, and the isopentenyl diphosphate isomerase gene of E. coli MG1655, including incorporated ribosome binding sites and flanking restriction endonuclease sites used in subsequent cloning steps.

FIG. 25 shows a cloning strategy for the production of plasmid pGB 1026.

FIG. 26 shows a cloning strategy for the production of plasmid pGB1033.

FIG. 27 shows a cloning strategy for the production of plasmid pGB1036.

FIG. 28 shows a gas chromatogram of a 1 ml sample of headspace of a 20 ml vial containing E. coli BL21 harboring plasmids pJ401-NEST-idi and pGB1036 cultured for 24 hours in Luria Bertani broth supplemented with 50 μg/ml kanamycin, 20 μg/ml chloramphenicol, and 200 μg/ml anhydrotetracycline. Peak 1 is linalool, with a retention time of 8.8 minutes. Peak 2 is 2-methyl-3-buten-2-ol, with a retention time of 4.8 minutes. The peak at 4.75 minutes has been identified as 2-butanone.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and/or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that comprise more than one unit unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

As used herein, the term “non-naturally occurring” when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a glycerol dissimilation or isoprene biosynthetic pathway. As defined herein, an “isoprene biosynthetic pathway” comprises a pathway, e.g., a series of one or more enzymes or activities involved in the production of isoprene by an organism, i.e., biologically, wherein one or more of those enzymes or activities is exogenous to the organism.

A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides or, functional fragments thereof. Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.

As used herein, the terms “microbe,” “microbial,” “microbial organism” or “microorganism” is intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.

“Exogenous” as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.

The non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refer to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as Escherichia coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or non-orthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if the proteins that they code for share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5′-3′ exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by structure or ancestry but with different functions. These might arise by, for example, duplication of a gene followed by evolutionary divergence to produce proteins with similar or common, but not identical functions. Paralogs can originate or derive from the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.

A non-orthologous gene displacement is a non-orthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a non-orthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires at least some structural similarity in the active site or binding region of a non-orthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, examples of non-orthologous genes include paralogs or unrelated genes.

Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having an isoprene biosynthetic pathway, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or non-orthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. Orthologs, paralogs and non-orthologous gene displacements can be determined by methods well known to those skilled in the art. As defined herein, enzymes or genes that are described or claimed as being “derived from” an organism include any homologs, paralogs, non-orthologous gene displacements that have substantially similar activity.

The methods and techniques utilized for culturing or generating the microorganisms disclosed herein are known to the skilled worker trained in microbiological and recombinant DNA techniques. Methods and techniques for growing microorganisms (e.g., bacterial cells), transporting isolated DNA molecules into the host cell and isolating, cloning and sequencing isolated nucleic acid molecules, knocking out expression of specific genes, etc., are examples of such techniques and methods. These methods are described in many items of the standard literature, which are incorporated herein in their entirety: “Basic Methods In Molecular Biology” (Davis, et al., eds. McGraw-Hill Professional, Columbus, Ohio, 1986); Miller, “Experiments in Molecular Genetics” (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1972); Miller, “A Short Course in Bacterial Genetics” (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1992); Singer and Berg, “Genes and Genomes” (University Science Books, Mill Valley, Calif., 1991); “Molecular Cloning: A Laboratory Manual,” 2^(nd) Ed. (Sambrook, et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989); “Handbook of Molecular and Cellular Methods in Biology and Medicine” (Kaufman, et al., eds., CRC Press, Boca Raton, Fla., 1995); “Methods in Plant Molecular Biology and Biotechnology” (Glick and Thompson, eds., CRC Press, Boca Raton, Fla., 1993); and Smith-Keary, “Molecular Genetics of Escherichia coli” (The Guilford Press, New York, N.Y., 1989).

Although the direct conversion of dimethylallyl diphosphate to isoprene by isoprene synthase enzymes is known in the art, we have shown that isoprene can also be produced from two different alcohols, 3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol, using linalool dehydratase-isomerase (see Example 1 and Example 2 and FIGS. 6 and 8), an enzyme isolated from Castellaniella defragrans strain 65Phen. The linalool dehydratase isomerase permits the development of novel isoprene biosynthetic pathways of either two or three steps. In a two-step isoprene biosynthetic pathway, dimethylallyl diphosphate is converted to 2-methyl-3-buten-2-ol by an enzyme such as a 2-methyl-3-buten-2-ol synthase, followed by conversion of 2-methyl-3-buten-2-ol to isoprene. In a three-step isoprene biosynthetic pathway, dimethylallyl diphosphate is converted to 3-methyl-2-buten-1-ol by either a phosphatase or a terpene synthase capable of converting dimethylallyl diphosphate to 3-methyl-2-buten-1-ol, 3-methyl-2-buten-1-ol is converted to 2-methyl-3-buten-2-ol by a 2-methyl-3-buten-2-ol isomerase, and 2-methyl-3-buten-2-ol is converted to isoprene by a 2-methyl-3-buten-2-ol dehydratase. As demonstrated in Example 1 and Example 2, the Castellaniella defragrans linalool dehydratase-isomerase functions as both a 2-methyl-3-buten-2-ol isomerase and a 2-methyl-3-buten-2-ol dehydratase.

Both the three-step isoprene biosynthetic pathway and the two-step isoprene biosynthetic pathway are expressed at a sufficient level to produce isoprene in detectable quantities. The isoprene may be detected and characterized by gas chromatography/mass spectrometry, for example.

Three-Step Isoprene Biosynthetic Pathway

As used herein, enzyme names are defined as follows. A 2-methyl-3-buten-2-ol dehydratase is an enzyme that catalyzes the conversion of 2-methyl-3-buten-2-ol to isoprene. A 2-methyl-3-buten-2-ol synthase is an enzyme that catalyzes the conversion of dimethylallyl diphosphate to 2-methyl-3-buten-2-ol. A 3-methyl-2-buten-1-ol synthase or prenol synthase is an enzyme that catalyzes the conversion of dimethylallyl diphosphate to 3-methyl-2-buten-1-ol also referred to herein as prenol. A 2-methyl-3-buten-2-ol isomerase is an enzyme that catalyzes the isomerization of 3-methyl-2-buten-1-ol to 2-methyl-3-buten-2-ol.

In one embodiment of the present invention, a non-naturally occurring microorganism containing one or more exogenous genes encoding enzymes of an isoprene biosynthetic pathway convert dimethylallyl diphosphate to isoprene in three steps (as used herein, “three-step isoprene biosynthetic pathway,” FIG. 1). In a first step, dimethylallyl diphosphate is converted to 3-methyl-2-buten-1-ol by a 3-methyl-2-buten-1-ol synthase. In a second step, 3-methyl-2-buten-1-ol is converted to 2-methyl-3-buten-2-ol by a 2-methyl-3-buten-2-ol isomerase. In a third step, 2-methyl-3-buten-2-ol is converted to isoprene by a 2-methyl-3-buten-2-ol dehydratase.

In a preferred embodiment of a naturally occurring microorganism for the conversion of dimethylallyl diphosphate to isoprene in three steps, the first step is catalyzed by a 3-methyl-2-buten-1-ol synthase and the second and third steps are catalyzed by a single, bi-functional enzyme with both 2-methyl-3-buten-2-ol isomerase and 2-methyl-3-buten-2-ol dehydratase activities.

The conversion of dimethylallyl diphosphate to 3-methyl-2-buten-1-ol (prenol) may be catalyzed by a phosphatase. Examples of such phosphatases include enzymes encoded by the Bacillus subtilis genes yqkG (nudF) and yhfR (Withers, S. T., Gottlieb, S. S., Lieu, B., Newman, J. D. and J. D. Keasling. 2007. Identification of isopentenol biosynthetic genes from Bacillus subtilis by a screening method based on isoprenoid precursor toxicity. Appl. Env. Microbiol. 73: 6277-6283), although other known phosphatases and coding sequences with predicted phosphatase activity, for example the ytjC gene of E. coli, may be used. Table 1, below, provides examples of phosphatases for use in the conversion of dimethylallyl diphosphate to prenol.

TABLE 1 GenBank Locus Accession No. Organism BAA12639 (YqkG) BAA12639 Bacillus subtilis CAA74541 (YhfR) CAA74541 Bacillus subtilis subsp. subtilis Strain 168 GPMB_ECOLI P0A7A2 Escherichia coli K-12 Calf Intestine Alkaline Phosphatase Shrimp Alkaline Phosphatase

The conversion of dimethylallyl diphosphate to 3-methyl-2-buten-1-ol may be catalyzed by a terpene synthase, e.g., a geraniol synthase or farnesol synthase or mutants thereof, for example. Table 2, below, provides examples of terpene synthases for use in the conversion of dimethylallyl diphosphate to 3-methyl-2-buten-1-ol.

TABLE 2 GenBank Locus Accession No. Organism Geraniol Synthase AAR11765 AAR11765 Ocimum basilicum ABB30216 ABB30216 Perilla citriodora ABB30217 ABB30217 Perilla citriodora ABB30218 ABB30218 Perilla frutescens CAE52821 CAE52821 Cinnamomum tenuipile Farnesol Synthase ACSS_MAIZE Q84ZW8 Zea mays ABJ16554 ABJ16554 Oryza sativa

3-methyl-2-buten-1-ol is isomerized to 2-methyl-3-buten-2-ol by a 2-methyl-3-buten-2-ol isomerase. As used herein, a 2-methyl-3-buten-2-ol isomerase is an enzyme that converts 3-methyl-2-buten-1-ol (prenol) to 2-methyl-3-buten-2-ol in a reversible reaction. An example of such an enzyme is the linalool dehydratase-isomerase of Castellaniella defragrans strain 65Phen, GenBank accession number FR669447. This enzyme catalyzes the isomerization of 3-methyl-2-buten-1-ol to 2-methyl-3-buten-2-ol and the dehydration of 2-methyl-3-buten-2-ol to isoprene (Example 1, below, and FIG. 6). Orthologs, paralogs and non-orthologous gene displacements of linalool dehydratase-isomerase can be determined by methods well known to those skilled in the art.

As used herein, a 2-methyl-3-buten-2-ol dehydratase is an enzyme that converts 2-methyl-3-buten-2-ol to isoprene. An example of such an enzyme is the linalool dehydratase-isomerase of Castellaniella defragrans strain 65Phen, GenBank accession number FR669447. This enzyme is capable of catalyzing the dehydration of 2-methyl-3-buten-2-ol to isoprene (Example 2, below, and FIG. 8). Orthologs, paralogs and non-orthologous gene displacements of linalool dehydratase-isomerase can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence that can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity that is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.

Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and the following parameters: Match: 1; mismatch: −2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

2-methyl-3-buten-2-ol dehydratase enzyme activity has also been identified in Aquincola tertiaricarbonis (Schuster, J., Schäfer, F., Hübler, N., Brandt, A., Rosell, M., Härtig, C., Harms, h., Müller, R. H. and T. Rohwerder. 2012. Bacterial degradation of tert-amyl alcohol proceeds via hemiterpene 2-methyl-3-buten-2-ol by employing the tertiary alcohol desaturase function of the Rieske nonheme mononuclear iron oxygenase MdpJ. J. Bact. 194: 972-981). The sequence of this 2-methyl-3-buten-2-ol dehydratase has not been reported.

Two-Step Isoprene Biosynthetic Pathway

In another embodiment, a non-naturally occurring microorganism containing one or more exogenous genes encoding enzymes of an isoprene biosynthetic pathway convert dimethylallyl diphosphate to isoprene in two steps catalyzed by a 2-methyl-3-buten-2-ol synthase (MBO synthase) and a 2-methyl-3-buten-2-ol dehydratase (as used herein, “two-step isoprene biosynthetic pathway,” FIG. 2).

As used herein, an example of a 2-methyl-3-buten-2-ol synthase is a naturally occurring polypeptide found in some plant plastids, particularly in the chloroplast, that converts dimethylallyl diphosphate to 2-methyl-3-buten-2-ol, and derivatives (mutants) of polypeptides that naturally convert dimethylallyl diphosphate to 2-methyl-3-buten-2-ol. MBO synthases are characterized, in part, by an amino-terminal plastid targeting sequence that routes the polypeptide to the chloroplast. Upon translocation into the chloroplast, the transit peptide may be cleaved from the polypeptide to yield a mature protein that is smaller in molecular weight than the precursor protein. For overexpression of an exogenous MBO synthase in a microbial organism, it is preferable to express a truncated MBO synthase that approximates the mature form found in nature, rather than the precursor form. Essentially, the sequence encoding the transit peptide is removed from the MBO synthase coding sequence. While visual inspection may allow one skilled in the art to select where to truncate the isoprene synthase coding sequence, computer-based algorithms such as ChloroP 1.1 can be used to help predict which amino acids belong to the transit peptide (Emanuelsson, 0., Nielsen, H., G. von Heijne. 1999. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci. 8: 978-984). An example of an MBO synthase is found in Pinus sabiniana, with the GenBank accession number AEB53064.1.

The conversion of dimethylallyl diphosphate to 2-methyl-3-buten-2-ol may be catalyzed by a terpene synthase, e.g., a linalool synthase (e.g. E.C. No. 4.2.3.25 or 4.2.3.26) or nerolidol synthase or mutants thereof, for example. Table 3, below, provides examples of terpene synthases for use in the conversion of dimethylallyl diphosphate to 2-methyl-3-buten-2-ol.

TABLE 3 GenBank Locus Accession No. Organism S-Linalool Synthase LIS_CLABR Q96376 Clarkia breweri LINS_ARATH Q84UV0 Arabidopsis thaliana C0KWV3_9LAMI C0KWV3 Perilla setoyensis C0KWV5_PERFR C0KWV5 Perilla frutescens var. hirtella C0KWV7_PERFR C0KWV7 Perilla frutescens var. hirtella D4N3A0_9ERIC D4N3A0 Actinidia arguta D4N3A1_9ERIC D4N3A1 Actinidia polygama R-Linalool Synthase LLOS1_ARTAN Q9SPN0 Artemesia annua LLOS_OCIBA Q5SBP3 Ocimum basilicum LLOS5_ARTAN Q9SPN1 Artemesia annua LLOS_MENAQ Q8H2B4 Mentha aquatica Q1XBU5_SOLLC Q1XBU5 Solanum lycopersicum (3S,6E)-Nerolidol Synthase Q5UB06_MEDTR Q5UB06 Medicago trunculata F8TWD1_POPTR F8TWD1 Populus trichocarpa NES1_FRAVE P0CV96 Fragaria vesca NES1_FRAAN P0CV94 Fragaria ananassa NES2_FRAAN P0CV95 Fragaria ananassa One example of the use of a terpene synthase to convert dimethylallyl diphosphate to 2-methyl-3-buten-2-ol is found in Example 3.

The conversion of 2-methyl-3-buten-2-ol to isoprene may be catalyzed by a 2-methyl-3-buten-2-ol dehydratase as described above. The 2-methyl-3-buten-2-ol dehydratase may be a bi-functional enzyme with both 2-methyl-3-buten-2-ol isomerase and 2-methyl-3-buten-2-ol dehydratase activities, such as the linalool dehydratase-isomerase described above, or the enzyme may encode only the 2-methyl-3-buten-2-ol dehydratase activity without a 2-methyl-3-buten-2-ol isomerase activity.

In a preferred embodiment of the present invention, dimethylallyl diphosphate available for conversion to isoprene by either a two-step isoprene biosynthetic pathway or a three-step isoprene biosynthetic pathway may be increased by overexpression of one or more endogenous genes or expression of one or more exogenous genes encoding enzymes of the methylerythritol phosphate pathway: 1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase, dimethylallyl-diphosphate/isopentenyl-diphosphate:NAD(P)⁺ oxidoreductase, or isopentenyl diphosphate isomerase. For example, expression of exogenous genes encoding 1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and isopentenyl diphosphate isomerase may result in increased levels of dimethylallyl diphosphate, and when expressed in conjunction with either a two-step isoprene biosynthetic pathway or a three-step isoprene biosynthetic pathway, result in increased yields of isoprene.

In another preferred embodiment of the present invention, dimethylallyl diphosphate available for conversion to isoprene by either a two-step isoprene biosynthetic pathway or a three-step isoprene biosynthetic pathway may be increased by expression of one or more exogenous genes encoding enzymes of the mevalonate pathway including, but not limited to: acetyl-CoA acetyltransferase (also known as thiolase), 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase. One example of overexpression of exogenous genes encoding the mevalonate pathway is described in Example 4.

In an additional embodiment, the present invention provides for a method of producing isoprene, the method comprising the steps of culturing a non-naturally occurring microbial organism comprising at least one or more exogenous nucleic acids encoding one or more enzymes of an isoprene biosynthetic pathway, wherein the one or more enzymes of an isoprene biosynthetic pathway are expressed in sufficient amounts to produce isoprene, in a suitable culture medium containing a carbon source under conditions such that the non-naturally occurring microorganism converts at least a part of the carbon source to isoprene, and recovering the isoprene. The carbon source may be or comprise glycerol, glucose, xylose, arabinose, or mixtures thereof; dimethylallyl diphosphate, 3-methyl-2-buten-1-ol, or 2-methyl-3-buten-2-ol. Preferably, the carbon source is or comprises glycerol, glucose or sugars derived from cellulosic biomass processes. The isoprene may be recovered as described in the examples below.

In the following examples of embodiments of the current invention, the common E. coli strain BL21 was used for the examples. BL21 (Life Technologies, Inc., Carlsbad, Calif.) cells were made electrocompetent and electroporated following the protocol from the MicroPulser Electroporation Apparatus Operating Instructions and Applications Guide (Bio-Rad catalog number 165-2100), except that LB without salt was used to grow up the culture in making cells electrocompetent.

Example 1 Microorganism for the Production of Isoprene from 3-methyl-2-buten-1-ol

This working example shows the production of isoprene from 3-methyl-2-buten-1-ol by a non-naturally occurring microorganism expressing one or more exogenous genes of an isoprene biosynthetic pathway.

The plasmid pJ404-LDI was constructed by DNA2.0 (Menlo Park, Calif.) using the E coli-codon-optimized sequence (SEQ ID NO: 1) of the linalool dehydratase-isomerase (LDI) of Castellaniella defragrans strain 65Phen. The LDI coding sequence was codon-optimized for expression in E. coli, synthesized and inserted into the plasmid expression vector pJexpress404. The resulting plasmid, pJ404-LDI, was electroporated into E. coli BL21 electrocompetent cells.

Plasmid pJ404-SAAT was constructed by DNA2.0 (Menlo Park, Calif.) using the codon-optimized sequence (SEQ ID NO: 2) of the strawberry acyl-CoA transferase (SAAT). The SAAT coding sequence was codon-optimized for expression in E. coli, synthesized and inserted into the plasmid expression vector pJexpress404. The resulting plasmid, pJ404-SAAT, was electroporated into E. coli BL21 electrocompetent cells. pJ404-SAAT was used as a negative control.

Transformants of BL21 harboring either pJ404-LDI or pJ404-SAAT were selected on Luria-Bertani (LB)-agar plates (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride, 15 g/L Bacto Agar) containing 100 μg/ml ampicillin.

A single colony of BL21 harboring pJ404-LDI or pJ404-SAAT from the LB-agar plates was used to inoculate 10 ml of LB broth (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride) containing 100 μg/ml ampicillin contained in 125-mL Erlenmeyer flasks. Flasks were incubated for 16 hours at 37° C. in a rotary shaking incubator. After 16 hours, the resulting cultures were diluted using fresh LB broth containing 100 μg/ml ampicillin to an optical density of 0.16 at 600 nm. 50 ml of the diluted cultures were placed in 300-ml Erlenmeyer flasks and incubated at 37° C. in a rotary shaking incubator until the optical density at 600 nm reached approximately 0.6, typically 90 minutes. 4 ml of the resulting cultures were then placed into 20 ml gas chromatography headspace vials. 3-methyl-2-buten-1-ol was added to a final concentration of 1 mM, IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to 0.1 mM, and the cultures were grown for an additional 16 hours at 37° C. with shaking.

Isoprene was measured using headspace analysis on an Agilent 7890A GC equipped with a CTC-PAL autosampler and a FID. Headspace vials (20 ml) were incubated at 50° C. with agitation at 500 rpm for 2 minutes. Then 1 ml of the headspace was removed using a heated headspace syringe at 50° C. and injected into the GC inlet (250° C., split of 20:1). Samples were analyzed using a FID detector set at 300° C., with a helium carrier gas flow rate of 2 ml/min through a DB-624 30 m×530 μm×3 μm column (J&W Scientific), and an oven program of 85° C. for 5.25 minutes. The isoprene concentration in samples was calculated from calibration curves generated from isoprene calibration gas standards analyzed under the same GC/FID method. The isoprene product was also confirmed by headspace GC/MS using an Agilent 7890A GC equipped with a 5975C MSD and a CTC-PAL autosampler. Headspace vials were incubated at 85° C. with agitation at 600 rpm for 5 minutes. Then 1 ml of the headspace was removed using a heated headspace syringe at 85° C. and injected into the GC inlet (250° C., split of 25:1). The GC/MS method used helium as the carrier gas at 1 ml/min through a HP-5MS 30 m×250 μm×0.25 μm column (J&W Scientific), an oven program of 35° C. for 4 minutes, then ramped 25° C./min to 150° C., a MS source temperature of 230° C., and a quadrupole temperature of 150° C. The mass spectrometer was operated in scan mode from 25 to 160 mass units. The isoprene peak was identified by the NIST 11 MS Library, as well as comparison against an authentic sample (135 ppm isoprene, 135 ppm carbon dioxide in dry nitrogen gas, Matheson TRIGAS, Houston, Tex.).

3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol were measured using headspace analysis on an Agilent 7890A GC equipped with a CTC-PAL autosampler and a FID. Headspace vials (20 ml) were incubated at 85° C. with agitation at 600 rpm for 5 minutes. Then 1 ml of the headspace was removed using a heated headspace syringe at 85° C. and injected into the GC inlet (250° C., split of 25:1). Samples were analyzed using a FID detector set at 350° C., with a helium carrier gas flow rate of 3 ml/min through at DB-624 30 m×530 μm×3 μm column (J&W Scientific), and an oven program of 90° C., then ramping 20° C./min to 230° C. for 3 minutes. The 3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol concentrations in samples were calculated from calibration curves generated from diluted standards of each compound analyzed under the same GC/FID method.

The results of this example are presented in FIG. 5 and FIG. 6. LB broth containing 1 mM 3-methyl-2-buten-1-ol without E. coli cells showed a peak at 3.96 minutes corresponding to 3-methyl-2-buten-1-ol (FIG. 5). Similarly, cultures containing 1 mM 3-methyl-2-buten-1-ol with BL21 cells harboring pJ404-SAAT showed a peak at 3.96 minutes corresponding to 3-methyl-2-buten-1-ol, and an additional peak corresponding to the aldehyde 3-methyl-2-buten-1-al (prenal, data not shown). In contrast, cultures containing 1 mM 3-methyl-2-buten-1-ol with BL21 cells harboring pJ404-LDI converted 3-methyl-2-buten-1-ol to 2-methyl-3-buten-2-ol and isoprene, corresponding to peaks at 2.96 minutes and 2.49 minutes, respectively. This demonstrates that E. coli cells harboring pJ404-LDI isomerize 3-methyl-2-buten-1-ol to 2-methyl-3-buten-2-ol and dehydrate 2-methyl-3-buten-2-ol to isoprene. FIG. 9 presents the GC/MS analysis of an authentic isoprene sample; FIG. 10 presents the GC/MS analysis of the peak with a 2.49 minute retention time, with the same fragmentation pattern as authentic isoprene shown in FIG. 9.

Example 2 Microorganism for the Production of Isoprene from 2-methyl-3-buten-2-ol

This working example shows the production of isoprene from 2-methyl-3-buten-2-ol by a non-naturally occurring microorganism expressing one or more exogenous genes of an isoprene biosynthetic pathway.

A single colony of BL21 harboring pJ404-LDI or pJ404-SAAT from LB-agar plates was used to inoculate 10 ml of LB broth (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride) containing 100 μg/ml ampicillin contained in 125-mL Erlenmeyer flasks. Flasks were incubated for 16 hours at 37° C. in a rotary shaking incubator. After 16 hours, the cultures were diluted using fresh LB broth containing 100 μg/ml ampicillin to an optical density of 0.16 at 600 nm. 50 ml of the diluted cultures were placed in 300-mL Erlenmeyer flasks and incubated at 37° C. in a rotary shaking incubator until the optical density at 600 nm reached approximately 0.6, typically 90 minutes. 4 ml of the cultures were then placed into 20-ml gas chromatography headspace vials. 2-methyl-3-buten-2-ol was added to a final concentration of 1 mM. IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 0.1 mM. Cultures containing 2-methyl-3-buten-2-ol were grown for 16 hours at 37° C. with shaking.

Isoprene, 3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol were measured as above. The identity of the isoprene peak was also verified using GC/MS, as described above in Example 1.

The results of this example are presented in FIG. 7 and FIG. 8. LB broth containing 1 mM 2-methyl-3-buten-2-ol with E. coli cells omitted showed a peak at 2.96 minutes corresponding to 2-methyl-3-buten-2-ol (FIG. 7). Similarly, cultures containing 1 mM 2-methyl-3-buten-2-ol and BL21 cells harboring pJ404-SAAT showed a peak at 2.96 minutes corresponding to 2-methyl-3-buten-2-ol (data not shown). In contrast, cultures containing 1 mM 2-methyl-3-buten-2-ol and BL21 cells harboring pJ404-LDI converted 2-methyl-3-buten-2-ol to isoprene, corresponding to the peak at 2.49 minutes. This demonstrates that E. coli cells harboring pJ404-LDI dehydrate 2-methyl-3-buten-2-ol to isoprene.

Example 3 FaNES1 Catalyzes Formation of 2-methyl-3-buten-2-ol from Dimethylallyl Diphosphate

This working example shows the production of 2-methyl-3-buten-2-ol from dimethylallyl diphosphate by a non-naturally occurring microorganism expressing an exogenous terpene synthase, the (3S,6E)-nerolidol synthase of Fragaria ananassa.

The plasmid pJ401-NEST-idi was constructed by DNA2.0 (Menlo Park, Calif.) using the codon-optimized sequence of the (3S,6E)-nerolidol synthase, FaNES1, of Fragaria ananassa(GenBank accession no. P0CV94; Aharoni, A., Giri, A. P., Verstappen, F. W. A., Bertea, C. M., Sevenier, R., Sun, Z., Jongsma, M. A., Schwab, W. and H. J. Bouwmeester. 2004. Gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species. The Plant Cell 16: 3110-3131) and the codon-optimized isopentenyl diphosphate isomerase gene, idi, of H. pluvialis. Both the FaNES1 and idi coding sequences were codon-optimized for expression in E. coli, synthesized and inserted into the plasmid expression vector pJexpress401. The resulting plasmid, pJ401-NES1-idi, was electroporated into E. coli BL21 electrocompetent cells. The codon-optimized sequence (SEQ ID NO: 4), including artificial ribosomal binding sites and flanking restriction endonucleases for subcloning, is provided in FIG. 12.

The production of nerolidol, linalool and 2-methyl-3-buten-2-ol was assayed as follows. A single colony of BL21 harboring plasmid pJ401-NES1-idi from an LB-agar plate was used to inoculate 10 ml of LB broth (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride) containing 50 μg/ml kanamycin. Flasks were incubated for 16 hours at 37° C. in a rotary shaking incubator. After 16 hours, the cultures were diluted using fresh LB broth containing 50 μg/ml kanamycin and 0.1 mM IPTG to yield an initial cell density at 600 nm of 0.4 to 0.5. 4 mL of the diluted culture was placed in a 20 ml GC vial and incubated for 6 or 24 hours at 30° C. with shaking. At 6 or 24 hours, the headspace gas was analyzed by GC/MS-SIM.

Samples were analyzed by headspace GC/MS in Select Ion Mode (SIM) using an Agilent 7890A GC equipped with a 5975C MSD and a CTC-PAL autosampler. Headspace vials (20 ml) were incubated at 85° C. with agitation at 600 rpm for 5 minutes. Then 1 ml of the headspace was removed using a heated headspace syringe at 85° C. and injected into the GC inlet (250° C., split of 25:1). Helium was used as the carrier gas at 1.5 ml/min through a VF-624MS 60 m×250 μm×1.4 μm column (J&W Scientific) and an oven program of 90° C. for 1 minute, then ramped 25° C./min to 230° C. for 5 min. The mass spectrometer was operated in SIM mode. The MS source temperature was 230° C., the quadrupole temperature was 150° C., and the solvent delay was 3.55 min. Concentrations of target analytes were determined from calibration curves of each analyte. Calibration standards for 2-methyl-3-buten-2-ol, 3-methyl-2-buten-1-ol, 3-methyl-2-butenal, and linalool were prepared in 10 mL of deionized water at concentrations of 1, 10, and 100 ppm. The headspace for each calibration standard was analyzed using the same GC/MS-SIM method. Isoprene was calibrated from certified gas standards at 14, 135, and 1375 ppm. Linear correlation coefficients for calibration curves were ≧0.99 for all impurity components. FIG. 13 shows a gas chromatogram of authentic linalool acquired under the GC/MS-SIM conditions. FIG. 14 shows a gas chromatogram of authentic 2-methyl-3-buten-2-ol acquired under the GC/MS-SIM conditions.

The results of this example are presented in FIG. 15. BL21 cells harboring pJ401-NES1-idi produced 1.47 mg/L linalool, corresponding to the peak at 8.8 minutes, and 0.05 mg/L 2-methyl-3-buten-2-ol, corresponding to the peak at 4.8 minutes. This demonstrates that E. coli cells harboring pJ401-NEST-idi produce 2-methyl-3-buten-2-ol in addition to linalool. The peaks corresponding to linalool and 2-methyl-3-buten-2-ol are absent from control cultures of BL21 harboring pJ404-SAAT grown under similar conditions (FIG. 16). The peaks were identified by the NIST 11 MS Library. The peaks for linalool (FIG. 17 and FIG. 18) and 2-methyl-3-buten-2-ol (FIG. 19 and FIG. 20) were also identified by comparison of retention times and ion fragmentation patterns against authentic samples.

Example 4 Overexpression of Mevalonate Pathway to Improve 2-methyl-3-buten-2-ol Production by FaNES1

This working example shows the production of 2-methyl-3-buten-2-ol from dimethylallyl diphosphate by a non-naturally occurring microorganism expressing an exogenous terpene synthase, the (3S,6E)-nerolidol synthase of Fragaria ananassa, can be enhanced by overexpression of a heterologous mevalonate pathway to increase the pool of dimethylallyl diphosphate available for conversion to 2-methyl-3-buten-2-ol.

The heterologous mevalonate pathway was constructed on a plasmid, pGB1036, as follows.

Plasmid pGA31R-MCS was constructed entirely by DNA synthesis, with the nucleotide sequence (SEQ ID NO: 5) presented in FIG. 21.

Plasmid pGS31R-MCS was constructed by replacing the p15A origin of replication on pGA31R-MCS with the low-copy pSC101 origin as an AvrII/SacI fragment using standard cloning techniques. The nucleotide sequence (SEQ ID NO: 6) is provided in FIG. 22.

Plasmid pJ248-mvaES was constructed using the codon-optimized sequence (SEQ ID NO: 7) of the mvaE and mvaS genes of Enterococcus faecalis ATCC 700802 (the codon-optimized sequences of mvaE and mvaS are as presented in FIG. 23). The mvaE and mvaS genes of Enterococcus faecalis ATCC 700802 were codon-optimized for expression in E. coli, synthesized and inserted in the plasmid pJ248. Unique ribosomal binding sites were included in front of each gene, along with flanking endonuclease restriction sites for use in plasmid construction.

Plasmid pJ241-MK.PMK.MPD.IDI containing a codon-optimized synthetic operon was constructed entirely by DNA synthesis, with the nucleotide sequence (SEQ ID NO: 8) presented in FIG. 24. The sequence of the synthetic operon, codon-optimized for expression in E. coli, encodes the mevalonate kinase gene of Methanocaldococcus jannaschi, the phosphomevalonate kinase of Enterococcus faecalis ATCC 700802, the mevalonate diphosphate decarboxylase of Saccharomyces cerevisiae S288C, and the isopentenyl diphosphate isomerase gene of E. coli MG1655, including incorporated ribosomal binding sites and flanking restriction endonuclease sites used in subsequent cloning steps.

Plasmid pGB 1008 was constructed by cloning the optimized mvaES genes from pJ248-mvaES into pGA31R-MCS as a KpnI/MluI DNA fragment using standard cloning techniques.

Plasmid pGB1026. The cloning strategy for pGB1026 is presented in FIG. 25. Plasmid pGB1026 was constructed by inserting an approximately 3,000 base pair PCR product encoding the pntAB genes of E. coli into the MluI site of pGB1008. The PCR product encoding the pntAB genes was amplified from genomic DNA of MG1655 using AccuPrime Pfx polymerase with the following oligonucleotide primers:

Primer 1: (SEQ ID NO: 9) 5′-CCG TAA CTA AAC GCG AAG GGA ATA TCA TGC GAA TTG G-3′ Primer 2: (SEQ ID NO: 10) 5′-CTA GAG ATC TAC GCG TCA GGG TTA CAG AGC TTT C-3′

Primer 1 incorporates a ribosomal binding site in front of the start codon of pntA. Primers 1 and 2 also include appropriate vector-overlapping 5′ sequences for use with the In-Fusion Advantage PCR Cloning Kit (Clontech). The PCR product was gel-purified, as was pGB1008 linearized with the restriction endonuclease MluI. Fragments were directionally joined together using the In-Fusion cloning kit and GC5 competent cells, following the manufacturer's directions. Transformants were screened, and the proper plasmid was identified through agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs.

Plasmid pGB1033 was created through the following process, illustrated in FIG. 26. pGB1026 was digested with the restriction endonucleases NcoI and SphI; the resulting 8.3 kb fragment was gel-purified. A second aliquot of pGB 1026 was digested with the restriction endonucleases MluI and SphI; the resulting 1.4 kb fragment was gel-purified. Plasmid pJ241-MK.PMK.MPD.IDI was digested with the restriction endonucleases NcoI and MluI; the resulting 4.1 kb containing the synthetic operon was gel-purified. The fragments were ligated together in a trimolecular ligation reaction using the NEB Quick Ligation Kit (New England BioLabs) and transformed into GC5 competent cells. Transformants were screened, and the proper plasmid was identified through agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs.

Plasmid pGB1036 was constructed by cloning the 2 operons from pGB1033, complete with promoters and terminators into pGS31R-MCS as a BamHI/AvrII DNA fragment using standard cloning techniques, as illustrated in FIG. 27. The fragments were ligated together using the NEB Quick Ligation Kit (New England BioLabs) and transformed into GC5 competent cells. Transformants were screened, and the proper plasmid was identified through agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs.

Plasmids pGB1036 and pJ401-NEST-idi, were co-transformed by electroporation into E. coli BL21 electrocompetent cells.

The production of nerolidol, linalool and 2-methyl-3-buten-2-ol was assayed as follows. A single colony of BL21 harboring plasmids pJ401-NES1-idi and pGB1036 from an LB-agar plate was used to inoculate 10 ml of LB broth (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride) containing 50 μg/ml kanamycin and 37 μg/ml chloramphenicol. Flasks were incubated for 16 hours at 37° C. in a rotary shaking incubator. After 16 hours, the cultures were diluted using fresh LB broth containing 50 μg/ml kanamycin, 20 μg/ml chloramphenicol, 200 μg/ml anydrotetracycline, and 0.1 mM IPTG to yield an initial cell density at 600 nm of 0.4 to 0.5. 4 ml of the diluted culture was placed in a 20 ml GC vial and incubated for 6 or 24 hours at 30° C. with shaking. At 6 or 24 hours, the headspace gas was analyzed by GC/MS-SIM as described in Example 3.

The results of this example are presented in FIG. 28. BL21 cells harboring pJ401-NEST-idi and pGB1036 produced approximately 4.05 mg/L linalool, corresponding to the peak at 8.8 minutes, and 0.38 mg/L 2-methyl-3-buten-2-ol, corresponding to the peak at 4.8 minutes. This demonstrates that E. coli cells harboring pJ401-NEST-idi and pGB1036 produce over 7 times more 2-methyl-3-buten-2-ol than cells harboring pJ401-NES1-idi by itself.

Example 5 FaNES 1 Mutations that Alter the Linalool to 2-methyl-3-buten-2-ol Ratio

This working example shows that the introduction of mutations into FaNES1 can alter the amount of linalool produced as compared to the amount of 2-methyl-3-buten-2-ol produced.

Site-directed mutagenesis or complete gene synthesis were used to introduce specific amino acid substitutions into the wild-type FaNES1 amino acid sequence. Table 4 presents the names of the mutant enzymes and the associated mutations. The amino acid numbering presented in Table 4 and this example correspond with the amino acid positions in the wild-type FaNES 1 enzyme as reported in GenBank accession no. P0CV94.

TABLE 4 Enzyme Name Introduced Mutation(s) NES1v2 I266F, S374F and I490F NES1#1 I266F NES1#2 S374F NES1#3 I490F NES1#4 G375D NES1#5 I266F and S374F NES1#6 I266F and 1490F NES1#7 S374F and I490F NES1#8 L413F NES1#9 I490K NES1#10 I490Y

NESv2 was produced from plasmid pJ401-NES1v2-idi using the codon-optimized sequence of the FaNES1 and the H. pluvialis idi genes. During construction, three amino acid mutations were introduced, converting the isoleucine at position 266 to phenylalanine (I266F), the serine at position 374 to phenylalanine (S374F), and the isoleucine at position 490 to phenylalanine (I490F).

FaNES1 mutants NES1#1 through NES1#10 were created through standard site-directed mutagenesis techniques using plasmid pJ401-NES1v2-idi as a template. The site-directed mutations were confirmed through DNA sequencing. Confirmed mutants were electroporated into E. coli BL21 electrocompetent cells. The production of linalool and 2-methyl-3-buten-2-ol for each individual mutant was assayed according to the methods described in Example 3, with a culture time of 6 hours at 30° C. The results are presented in Table 5.

TABLE 5 FaNES1 Variant Linalool 2-methyl-3-buten-2-ol Enzyme (mg/L) (mg/L) Wild-type 0.54 0.02 NESv2 — — NES#1 — — NES#2 — — NES#3 — — NES#4 — — NES#5 — — NES#6 — — NES#7 — — NES#8 0.05 — NES#9 — — NES#10 — —

Since it had been shown (Example 4) that increasing the supply of dimethylallyl diphosphate results in increased production of linalool and 2-methyl-3-buten-2-ol by wild-type FaNES1, a subset of the plasmids encoding FaNES1 variants were co-transformed with pGB 1036 into BL21 electrocompetent cells. The ability of the variant enzymes to produce linalool and 2-methyl-3-buten-2-ol was assayed according to the methods presented in Example 3, with a 24 hour incubation at 30° C. The results are presented in Table 6.

TABLE 6 FaNES1 Variant Enzyme* Lin* (mg/L) 232-MB* (mg/L) Wild-type 4.05 0.38 NES1#1 0.04 NES1#3 0.08 0.04 NES1#8 0.79 0.05 NES1#9 XX XX NES1#10 XX XX *In vivo assay performed with enhanced dimethylallyl diphosphate concentrations provided by pGB1036; Lin = Linalool; 232-MB = 2-methyl-3-buten-2-ol.

Example 6 Microorganism for the Production of Isoprene from Dimethylallyl Diphosphate

This example demonstrates how one may produce isoprene with a non-naturally occurring microorganism expressing a phosphatase, a 2-methyl-3-buten-2-ol isomerase, and a 2-methyl-3-buten-2-ol dehydratase.

The yhfR gene of Bacillus subtilis was codon-optimized for expression in E. coli, synthesized and inserted into the plasmid vector pJex404 to produce pJex404-yhfR. The codon-optimized yhfR sequence (SEQ ID NO: 3), including a ribosome binding site, is presented in FIG. 11. The ribosome binding site and yhfR coding sequence were amplified by polymerase chain reaction (PCR) using the following oligonucleotide primers:

(SEQ ID NO: 11) 5′-GGG CAA GTA ACT CGA TTA AAG AGG AGA AAA TAT  AAT GAC GGC AG-3′ (SEQ ID NO: 12) 5′-GCC CTT GGG GCT CGA GTT ATT TGA TGA AAC CGC  TCA GAT GG-3′

Plasmid pJ404-LDI was linearized by endonuclease restriction with the enzyme XhoI. The PCR product containing the yhfR coding sequence and the XhoI-digested pJ404-LDI were agarose gel-purified using standard laboratory techniques. The fragments were joined together using the In-Fusion Advantage PCR Cloning Kit (Clontech Laboratories, Inc., Mountain View, Calif.), then transformed into chemically competent E. coli GC5 cells (Gene Choice, available from Sigma-Aldrich Co. LLC) following the manufacturer's directions. Transformants were screened, and the proper plasmid was identified through agarose gel electrophoresis of restriction endonuclease-digested plasmid DNAs. The proper plasmid was then transformed into electrocompetent E. coli BL21. The resulting plasmid was designated pJ404-LDI.yhfR.

The production of isoprene by BL21 harboring plasmid pJ404-LDI.yhfR may be assayed as follows. A single colony of BL21 harboring pJ404-LDI.yhfR or pJ404-LDI from LB-agar plates are used to inoculate 10 mL of LB broth (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride) containing 20 g/L glycerol and 100 μg/ml ampicillin contained in 125 mL Erlenmeyer flasks. Flasks are incubated for 16 hours at 37° C. in a rotary shaking incubator. After 16 hours, the cultures are diluted using fresh LB broth containing 20 g/L glycerol and 100 μg/ml ampicillin to an optical density of 0.16 at 600 nm. 50 ml of the diluted cultures are placed in 300-mL Erlenmeyer flasks and incubated at 37° C. in a rotary shaking incubator until the optical density at 600 nm reaches approximately 0.6, typically 90 minutes. 4 ml of the cultures are then placed into 20 ml gas chromatography headspace vials. IPTG (Isopropyl β-D-1-thiogalactopyranoside) is added to 0.1 mM. Cultures are grown for 16 hours at 37° C. with shaking.

Isoprene, 3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol are measured as above. The identity of the isoprene peak may be verified using GC/MS, as described above.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While the foregoing is directed to embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. 

What is claimed is:
 1. A method of producing isoprene, the method comprising the steps of: a) optionally converting 3-methyl-2-buten-1-ol to 2-methyl-3-buten-2-ol using a 2-methyl-3-buten-2-ol isomerase enzyme; and b) converting 2-methyl-3-buten-2-ol to isoprene using a 2-methyl-3-buten-2-ol dehydratase enzyme.
 2. The method of claim 1, wherein the 2-methyl-3-buten-2-ol isomerase enzyme is expressed by an exogenous nucleic acid in a microbial organism.
 3. The method of claim 1, wherein the 2-methyl-3-buten-2-ol dehydratase enzyme is expressed by an exogenous nucleic acid in a microbial organism.
 4. The method of claim 1, wherein the 2-methyl-3-buten-2-ol dehydratase is a bi-functional enzyme further comprising 2-methyl-3-buten-2-ol isomerase activity.
 5. The method of claim 4, wherein the 2-methyl-3-buten-2-ol dehydratase is a linalool dehydratase-isomerase.
 6. The method of claim 5, wherein the linalool dehydratase-isomerase is derived from Castellaniella defragrans.
 7. The method of claim 5, wherein the linalool dehydratase-isomerase is expressed by an exogenous nucleic acid in microbial organism.
 8. The method of claim 2, wherein the microbial organism is selected from bacteria, archaea, eubacteria, yeast and fungi.
 9. The method of claim 8, wherein the microbial organism is an Escherichia coli.
 10. The method of claim 3, wherein the microbial organism is selected from bacteria, archaea, eubacteria, yeast and fungi.
 11. The method of claim 10, wherein the microbial organism is an Escherichia coli.
 12. The method of claim 7, wherein the microbial organism is an Escherichia coli. 